PROJECT SUMMARY The enzymatic modification of proteins through tyrosine phosphorylation is a common mechanism for relaying information in animal cells. Tyrosine kinases act in a signal-responsive manner to phosphorylate specific proteins at tyrosine residues, and the opposing tyrosine phosphatases dephosphorylate proteins to dynamically regulate signals. Tyrosine phosphorylation is essential to many biological processes in healthy cells, and the dysregulation of tyrosine phosphorylation is a common feature of many diseases, most notably cancers. Over the past few decades, we have developed an extensive understanding of tyrosine kinase function and regulation, but our knowledge of tyrosine phosphatases lags behind. This disparity is partly due to the fact that it is easier to develop drugs that target tyrosine kinases than tyrosine phosphatases, and the therapeutic potential of tyrosine kinases has motivated the development of robust tools to study their structure, biochemistry, and biology. The overarching goals of my lab are to understand, at the molecular level, how tyrosine phosphatases select substrate proteins to dephosphorylate, how they are regulated through dynamic changes in their structure, and how they contribute to healthy and disease-associated signaling. Over the next five years, my group will devise new techniques to study tyrosine phosphatases. We are currently developing a high-throughput biochemical platform to rapidly identify and compare the substrate sequence preferences of tyrosine phosphatases. These analyses will be conducted in parallel with proximity-labeling experiments in live cells to tag the interaction partners of tyrosine phosphatases for identification by mass spectrometry. Together, these approaches will allow us to map the substrates of tyrosine phosphatases and help define the signaling roles of these enzymes. We are also developing methods to rapidly characterize the functional effects of all possible point mutations in a tyrosine phosphatase. These mutational screens will allow us to identify new modes of regulation, pinpoint the functional consequences of disease-associated mutations, and map likely drug-resistance mutations that may arise to phosphatase-targeted cancer therapies. We are broadly interested in two areas of signaling biology: diseases where tyrosine phosphatases are mutated and/or dysregulated, and the activation of immune T cells. As we develop new biochemical tools, we will initially apply these tools to the tyrosine phosphatase SHP2, which plays a causal role in several congenital diseases and cancers, and is also critical to normal signaling in many cell types, including T cells. Our work will clarify the signaling functions of SHP2, connect known mutations to specific phenotypes, and help guide the development of SHP2-targeted therapies. In the long-term, we will apply our novel approaches to other tyrosine phosphatases.